Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Biocides; animal or insect repellents or attractants
Reexamination Certificate
1999-08-10
2001-11-13
Levy, Neil S. (Department: 1616)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Biocides; animal or insect repellents or attractants
C424S409000, C514S471000, C523S122000, C523S123000
Reexamination Certificate
active
06316014
ABSTRACT:
TECHNICAL SECTOR
The present invention is related to the branch of Microbiology and in particular with a microcidal composition which contains as active principle 1-(5-bromophur-2-yl)-2-bromo-2-nitroethene, which is used as chemical sterilizer of culture media for the control of bacterial and fungal contaminants.
PRIOR ART
The contamination of culture media for example, in the production of plants cultivated “in vitro”, cause substantial losses to the whole process since the autoclave only eliminates at-the-moment pollution. In some cases different antibiotics, e.g., Cefotaxim, Novobiocin, and Dihydrostreptomycin have been used to prevent contamination without satisfactory results.
The compound 1-(5-bromophur-2-yl)-2-bromo-2-nitroethenehas been previously described (Z. N. Nazarova, 1972. “Synthesis of some furylnitroolefins with potential biological activity, Khim. Farm. Zh. 6(10,) pp. 5-8 and Z. N. Nazarova et al, 1972.” Physicochemical properties and reactivity of furylnitroolefins, Zh, Org. Khim. 8 (2), pp. 404-11) and it is also known as a powerful agent with antibacterial and antifungal activities (European patent application No. EP-A 0 678 516).
DISCLOSURE OF THE INVENTION
The present invention provides a microcidal composition which can be advantageously used for the control of contaminants in culture media, such as bacterial or fungal contaminants.
This technical solution, even can be used for the sterilization of solid and liquid culture media, is particularly useful in the production of “in viro” cultivated plants, and surprisingly was found that its utilization can substitute completely the previous process of sterilization that this media require and even more, the addition of the necessary hormones in the rootage process of in vitro plants of crops.
The main limitation against the use of this active principle as sterilizer agent in solid media has been its close-to-null water solubility and the also reduced dissolution rate in an aqueous medium. A possible solution to this problem could be solid dispersions, which allow for a decrease of the particle size to molecular level.
The method proposed in the present provides a microcidal composition which contains as active principle 1-(5-bromophur-2-yl)-2-bromo-2-nitroethene as sterilizing agent and consists in dispersion by fusion or co-precipitation of this compound in a physiologically inert matrix and easily soluble in water, which is formed by a high molecular weight polyethylene glycol and other polymers.
In order to carry out the dispersion by fusion of the 1-(5-bromophur-2-yl)-2-bromo-2-nitroethene, the selection of the manufacture methods and temperature parameters, as well as that of stirring times were based on the melting point and thermal stability of the active principle and the polymer elected.
The compound 1-(5-bromophur-2-yl)-2-bromo-2-nitroethene is melted without decomposition at a temperature of 89-90° C. and is thermally stable up to 110° C.
However, the elaboration of solid disperse systems of the active principle with polymers whose melting points are higher than 110° C. affect the active principle thermal stability and, therefore, it is recommended the co-precipitation method in a solvent.
In order to carry out the dispersion by fusion of the 1-(5-bromophur-2-yl)2-bromo-2-nitroethene, physical mixture of the active principle and PEG-6000 in a ration from 1:20 to 1:1 heats up to fusion at the temperature of 60-80° C. and remains under constant stirring for 15 minutes until an entire homogeneity of the melting is reached. Then, the mixture melted is poured and spread as a thin layer on refrigerated stainless steel plates, thus ensuring the speed of the solidification process. The solid obtained is grinded in mills and screened at a particle size smaller than 0.2 mm.
The melting point range of solid dispersions of the active principle and PEG-6000 varies with the composition of the mixture, being 53-540° C. for the solid dispersion by fusion dispersion at 30% whose composition coincides with the eutectic composition of the binary system active principle-PEG-6000.
In order to obtain a dispersion by co-precipitation of 1-(5-bromophur-2-yl)-2-bromo-2-nitroethene the mixture of the active principle and the polymer in ratios from 1:20 to 1:1 is dissolved in a polar solvent heated in an interval of 10-60 minutes at a temperature ranging between 40 and 60° C. Then, it is vacuumrotoevaporated controlling temperature from 40-80° C. The solid thus obtained is grinded in a mill and screened at a particle size smaller than 0.2 mm
For the elaboration of liquid culture medium containing 1-(5-bromophur-2-yl)-2-bromo-2-nitroethene, the compound is included in a aqueous formulation using tensioactive compounds such as Solutol IIS-15 and Cremophor, among other compounds. This compositions has been prepared looking for a good stability and solubility of the compound and in a concentration that does not affect adversely the organisms which are cultivated in said medium.
Once they are obtained stable compositions containing 1-(5-bromophur-2-yl)-2-bromo-2-nitroethene using the methods described above, different assays were carry out in order to know the optimum dose, particularly using the compound as sterilizer agent of solid culture media used for the propagation of “in vitro” cultivated plants. Phytoxicity was evaluated considering necrosis, chlorosis, multiplication coefficient and junction-to-junction distance (for potatoes),
For those cultures studied (banana, potato and sugar cane), the optimum dose of the 1-(5-bromophur-2-yl)-2-bromo-2-nitroethene was in a range between 30 and 45 □g/ml.
REFERENCES:
patent: 4017627 (1977-04-01), Styrm
patent: 4965254 (1990-10-01), Anderson et al.
patent: 0 687 516 A (1995-10-01), None
patent: 57 038703 A (1982-03-01), None
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patent: WO 89 11793 (1989-12-01), None
Castañedo Cancio Nilo Ramón
de la Caridad Cueto Sánchez Madaysi
Diaz Martinez Grisell
González Bedia Mirtha Maira
González Lorenzo Carlos Manuel
Centro de Bioactivos Quimicos (CBQ)
Lackenbach Siegel
Levy Neil S.
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